Halohydrin epoxidase is also called halohydrin hydrogen-halide-lyase, halohydrin dehalogenase or haloalcohol dehalogenase, and is an enzyme having the activity to convert 1,3-dihalo-2-propanol into epihalohydrin and the activity to catalyze a reverse reaction thereof (EC number: 4.5.1.-). It is known that halohydrin epoxidase is roughly classified into 3 groups (Groups A, B and C) based on the amino acid sequence homology, etc. (Non-patent document 1: J. Bacteriology, 183 (17), 5058-5066, 2001). Examples of halohydrin epoxidases classified into Group A include HheA from Corynebacterium sp. strain N-1074 (Non-patent document 2: Biosci. Biotechnol. Biochem., 58 (8), 1451-1457, 1994), HheAAD2 from Arthrobacter sp. strain AD2 (Non-patent document 1), and Deh-PY1 from Arthrobacter sp. strain PY1 (Non-patent document 3: J. Health. Sci., 50 (6), 605-612, 2004). Examples of halohydrin epoxidases classified into Group B include HheB from Corynebacterium sp. strain N-1074 (Non-patent document 2). HheBGP1 from Mycobacterium sp. strain GP1 (Non-patent document 1), and DehA from Arthrobacter erithii strain H10a (Non-patent document 4: Enz. Microbiol. Technol., 22, 568-574, 1998). Examples of halohydrin epoxidases classified into Group C include HheC from Agrobacterium radiobacter strain DH094 (Patent document 1: Japanese Laid-Open Patent Publication No. 10-210981), HheC from Agrobacterium radiobacter strain AD1 (Non-patent document), and HalB from Agrobacterium tumefaciens (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=4960076#feature—4960076) (Non-patent document 5: Thesis (1996) University of Wales, Cardiff, United Kingdom).
Epihalohydrin is a useful substance as a raw material for synthesis of various pharmaceutical products and physiologically active substances. For example, it is known that (R)-(−)-4-halo-3-hydroxybutyronitrile, which is obtained by ring-opening cyanation of (R)-epihalohydrin, is useful as a raw material for synthesis of L-carnitine (Patent document 2: Japanese Laid-Open Patent Publication No. 57-165352). It has become clear that at least a part of halohydrin epoxidases have the activity to catalyze a reaction in which epihalohydrin is subjected to ring-opening cyanation in the presence of a cyanogen compound to produce 4-halo-3-hydroxybutyronitrile in addition to the above-described activity to convert 1,3-dihalo-2-propanol into epihalohydrin and the activity to catalyze a reverse reaction thereof. As examples of utilization of the reaction, methods for producing an optically-active 4-halo-3-hydroxybutyronitrile from 1,3-dihalo-2-propanol (Patent document 3: Japanese Laid-Open Patent Publication No. 3-053889; Patent document 4: Japanese Laid-Open Patent Publication No. 2001-25397) and a method for producing an optically-active 4-halo-3-hydroxybutyronitrile from epihalohydrin (Patent document 5: Japanese Laid-Open Patent Publication No. 3-053890) are known.
The halohydrin epoxidase activity per cell of a microorganism separated from nature is not necessarily high enough from the viewpoint of industrial utilization. In order to solve this problem, attempts have been made to improve the halohydrin epoxidase activity per transformant utilizing the genetic engineering technique. For example, it is indicated that optically-active 4-halo-3-hydroxybutyronitrile can be efficiently produced using various transformants obtained by introducing various expression plasmids, which are obtained by cloning a gene encoding halohydrin epoxidase HheB from Corynebacterium sp. strain N-1074 (Patent document 6: Japanese Laid-Open Patent Publication No. 4-278089; Patent document 7: Japanese Laid-Open Patent Publication No. 5-317066; and Patent document 8: Japanese Laid-Open Patent Publication No. 2007-049932). Using these transformants, many copies of halohydrin epoxidase gene are retained in a bacterium, thereby significantly improving the halohydrin epoxidase activity per transformant.
Meanwhile, because of the recent progress of the genetic engineering technique, it has become possible to intentionally prepare a mutant in which one or more constituent amino acids of enzyme protein are deleted, added, inserted, or substituted with other amino acids. It is known that such mutants may provide improved performances such as activity, stability, organic solvent resistance, heat resistance, acid resistance, alkali resistance and substrate specificity compared to an enzyme without mutation depending on the type of mutation. The improvement of these performances may effect significant reduction in production cost in industrial production utilizing enzyme reactions through reduction in production cost of enzyme catalyst per activity, stabilization of enzyme catalyst, simplification of reaction process, improvement of reaction yield, etc. Therefore, utilizing many enzymes, useful improved enzymes with various improved performances have been created. It has been reported that mutants of halohydrin epoxidase in which one or more constituent amino acids are deleted, added, inserted or substituted with other amino acids were produced. For example, Patent document 9 (International Publication WO 2005/017141 pamphlet) describes 369 types of mutants of HheC from Agrobacterium radiobacter strain AD1 and a mutant of HheB from Corynebacterium sp. strain N-1074. Patent document 10 (US Laid-Open Publication No. 2005/0272064) describes 570 types of mutants of HheC from Agrobacterium radiobacter strain AD1 and a mutant of HheB from Corynebacterium sp. strain N-1074. In addition, Patent document 11 (US Laid-Open Publication No. 2006/0099700) describes 1422 types of mutants of HheC from Agrobacterium radiobacter strain AD1 and a mutant of HheB from Corynebacterium sp. strain N-1074. It is indicated that the activity in the reaction in which ethyl (R)-4-chloro-3-hydroxybutyrate is converted into ethyl (S)-4-cyano-3-hydroxybutyrate was improved when using several of these mutants. Non-patent document 6 (J. Bacteriol., 183 (17), 5058-5066, 2001) describes Ser132Ala, Ser132Cys, Tyr145Phe, Arg149Lys, Arg149Glu and Arg149Gln, which are mutants of HheC from Agrobacterium radiobacter strain AD1. Non-patent document 7 (Enz. Microbiol., Technol. 30, 251-258, 2002) describes C30A, C153S, C229A and C153S/C229A, which are mutants of HheC from Agrobacterium radiobacter strain AD1. It is indicated in the document that the stability in the case of the mutants C30A and C153S was improved compared to that in the case of wild-type halohydrin epoxidase. Non-patent document 8 (EMBO J., 22 (19), 4933-4944, 2003) describes Asp80Asn and Asp80Ala, which are mutants of HheC from Agrobacterium radiobacter strain AD1. Non-patent document 9 (Biochemistry, 42 (47), 14057-14065, 2003) describes W139F, W192F, W238F and W249F, which are mutants of HheC from Agrobacterium radiobacter strain AD1. Non-patent document 10 (Biochemistry, 44 (17), 6609-6618, 2005) describes N176A, N176D and Y187F, which are mutants of HheC from Agrobacterium radiobacter strain AD1. All of these examples relate to mutants of halohydrin epoxidase HheC from Agrobacterium radiobacter strain AD1.    Patent document 1: Japanese Laid-Open Patent Publication No. 10-210981    Patent document 2: Japanese Laid-Open Patent Publication No. 57-165352    Patent document 3: Japanese Laid-Open Patent Publication No. 3-053889    Patent document 4: Japanese Laid-Open Patent Publication No. 2001-25397    Patent document 5: Japanese Laid-Open Patent Publication No. 3-053890    Patent document 6: Japanese Laid-Open Patent Publication No. 4-278089    Patent document 7: Japanese Laid-Open Patent Publication No. 5-317066    Patent document 8: Japanese Laid-Open Patent Publication No. 2007-049932    Patent document 9: International Publication WO 2005/017141 pamphlet    Patent document 10: US Laid-Open Publication No. 2005/0272064    Patent document 11: US Laid-Open Publication No. 2006/0099700    Non-patent document 1: J. Bacteriol., 183 (17), 5058-5066, 2001    Non-patent document 2: Biosci. Biotechnol. Biochem., 58 (8), 1451-1457, 1994    Non-patent document 3: J. Health. Sci. 50 (6), 605-612, 2004    Non-patent document 4: Enz. Microbiol. Technol., 22, 568-574, 1998    Non-patent document 5: Thesis (1996) University of Wales, Cardiff, United Kingdom    Non-patent document 6: J. Bacteriol., 183 (17), 5058-5066, 2001    Non-patent document 7: Enz. Microbiol., Technol. 30, 251-258, 2002    Non-patent document 8: EMBO J., 22 (19), 4933-4944, 2003    Non-patent document 9: Biochemistry, 42 (47), 14057-14065, 2003    Non-patent document 10: Biochemistry, 44 (17), 6609-6618, 2005